1. Field of the Invention
The present application relates to a method of regenerating nerve or attenuating degeneration of injured nerve by administering at or an area near an injured nerve, a nerve regenerating or nerve degeneration attenuating amount of phosphatase and tensin homolog (PTEN) lipid phosphatase inhibiting peptide.
2. General Background and State of the Art
In adult mammalian nervous system, regeneration of damaged neurons hardly occurs in healing response to nerve injury. There are two main reasons why adult CNS neurons fail to regenerate after injury—axons do not regenerate in adult central nervous system not only because of its inhibition by secreted extracellular inhibitory factors upon injury, but also because of the loss of intrinsic axon growth ability, which rapidly declines through aging [Schwab et al; 1996, Goldberg et al. 2002; Filbin et al. 2006; Fitch et. al 2008]. However, elimination of extracellular inhibitory molecules secreted upon nerve injury only triggers very limited axon regeneration in vivo [Yiu et. al 2006; Hellal et al. 2011]. Thus, promoting axonal regeneration process by regulation of intrinsic nerve outgrowth is currently focus of a therapeutic target for nerve injury treatment.
PTEN (phosphatase and tensin homolog) protein is a dual phosphatase and is considered to be important as tumor suppressor by negatively regulating phosphatidylinositol3-kinase (PI3K) signaling pathway. The PI3K signaling pathway is a critical signal transduction pathway for cell proliferation, survival and differentiation as well as protein synthesis, metabolism and motility [Zhang et al. 2010]. As a lipid phosphatase, PTEN catalyzes conversion of phosphatidylinositol (3,4,5) triphosphate (PIP3) to phosphatidylinositol (4,5) diphosphate (PIP2) by dephosphorylating the 3-position of PIP3, hence suppressing PI3K signaling pathway by antagonizing PI3K activity. [Di Cristofano et. al 2010]. Deletion or inactivation of PTEN enhances PI3K activity and promotes activation of downstream components of PI3K signaling pathway, including PDK1, Akt and mammalian target of rapamycin (mTOR), which leads to tumor formation [Di Cristofano et. al 2010; Stambolic et al. 1998].
Regulation of PI3K-mediated signaling by PTEN is also deeply related to nerve regeneration process in nerve system. Recent studies reveal that inhibition of PTEN protein or deletion of PTEN gene facilitates intrinsic regenerative outgrowth of adult CNS/PNS nerve upon Injury [Park et. al 2008; Liu et. al 2010; Sun et. al 2012; Christie et. al 2012]. For example, Park et al. found that deletion of PTEN in adult rat retinal ganglion cells (RGCs) using conditional knockout mice actually promotes robust axon regeneration after optic nerve injury by reactivating PI3K-Akt-mTOR signaling pathway. Reactivating mTOR pathway by conditional knockout of another negative regulator of the mTOR pathway also leads to axon regeneration, indicating that promotion of PI3K-mTOR signaling may be a key factor for restoring intrinsic axon regeneration ability. Also, Liu et al. reported that conditional deletion of PTEN in in vivo CNS injury model actually increases the diminished neuronal mTOR activity upon CNS injury by up-regulating PI3K signaling pathway, which leads to enhanced compensatory sprouting of uninjured CST axons and successful regeneration of injured CST axons past a spinal cord lesion. In case of PNS injury, inhibition of PTEN both in vitro and in vivo also increases axonal outgrowth [Christie et. al 2012]. Thus, developing PTEN inhibitor for promoting PI3K-mTOR signaling pathway is a good therapeutic target to enhance axon regeneration in injured nerve system. the PTEN inhibitor may be used in combined therapeutic methodology with existing or novel cell therapy containing other effective reagents for nerve regeneration after CNS or PNS injury.
In this study, we developed potential PTEN inhibitors effective for nerve regeneration and/or protection from nerve degeneration by stimulating PI3K signaling pathway. For activation of PTEN as lipid phosphatase, PTEN must localize in the plasma membrane in an appropriate orientation [Leslie et. al 2008]. Thus, we investigated the mechanism of PTEN membrane localization to design potential PTEN inhibitor candidates in peptide form. Three different peptides—TGN-1, TGN-2 and TGN-3—were designed and synthesized as potential PTEN inhibitors and their inhibitory ability against PTEN activity using in vitro PTEN activity assay was investigated. We also characterized their effect on regulation of PI3K signaling pathway by using neuronal cell lines. We discovered that TGN-1 and TGN-2 peptides, which are modified peptides mimicking the phosphorylation site in PTEN C-terminal region, actually diminished PTEN lipid phosphatase activity in in vitro PTEN activity assay. TGN-1 peptide also enhanced the activation level of Akt protein in PC12 cells, indicating that these peptides are effective to up-regulate PI3K-Akt signaling pathway. Neurite assay with neuronal cell showed that TGN-1 and TGN-2 peptides promoted neurite outgrowth as well as delayed neurite degeneration by enhancing neurite microtubule structure. Therefore, TGN peptides are useful as a therapeutic agent for nerve regeneration after CNS injury.